In thalassemia and sickle cell disease the severity of symptoms can be ameliorated by enhanced expression of the fetal gamma-globin genes in adult erythroid cells in which the gamma-genes are normally silenced. This application proposes to test the hypothesis that the intergenic DNA between the Agamma- and delta-globin genes including the psi/beta-globin gene enhancer and promoter and the Alu repetitive DNAs as well as the beta-globin promoter participates in the silencing of gamma-genes by competing with the gamma-promoter for binding with specific transcription factors (TFs) present in limited amounts in adult erythroid cells. Two new systems will be used for the study: in vitro culture of human erythroid progenitor CFU-E cells obtained from adult blood and transgenic (Tg) zebrafish harboring 100 kb of BAC DNA spanning the entire human beta-globin gene locus, in which the gamma-globin genes undergo silencing during erythroid differentiation and development. The specific aims are: (1). Identification of TFs that regulate gamma-globin gene inactivation and assess their relative abundance during human erythropoiesis from CFU-E to erythroblast cells by i) microarray analysis to identify known and also new and unknown TFs differentially expressed in CFU-E and erythroblast cells, ii) over-expressing these TFs in CFU-E cells to determine their activities on y-gene expression, iii) gel mobility shift assays with nuclear extracts of CFU-E and erythroblast cells to study the binding affinities of the TFs to cognate DNA motifs in the globin promoters and the intergenic DNAs, and iv) Chromatin immunoprecipitation (CHIP) to map in vivo changes in the assembly of these TFs into pol II or pol III transcriptional machinery in the beta-globin gene locus. 2). Identification of cis-DNA elements that regulate inactivation of human gamma-globin genes during ontogeny in Tg zebrafish. Interginic DNA and globin promoters will be deleted in vivo from the integrated human BAC DNA by Cre-loxP mediated recombination. The effects of the deletions on gamma-gene expression and on the assembly of the TFs into transcriptional machinery at the human globin gene locus will be studied by quantitative RTPCR and CHIP. Establishing and understanding the in vitro human adult erythropoiesis and Tg zebrafish models of gamma-globin gene silencing will provide new targets and new systems to test pharmacological compounds designed to regulated gamma-globin.